Description of a New Octoploid Frog Species

Journal of Zoology

Journal of Zoology. Print ISSN 0952-8369 Description of a new octoploid frog species (Anura: Pipidae: Xenopus) from the Democratic Republic of the Congo, with a discussion of the biogeography of African clawed frogs in the Albertine Rift B. J. Evans1, E. Greenbaum2, C. Kusamba3, T. F. Carter1, M. L. Tobias4, S. A. Mendel1 & D. B. Kelley4

1 Department of Biology, Center for Environmental Genomics, McMaster University, Hamilton, ON, Canada 2 Department of Biological Sciences, University of Texas at El Paso, El Paso, TX, USA 3 Laboratoire d’Herpe´ tologie, De´ partement de Biologie, Centre de Recherche en Sciences Naturelles, Lwiro, Re´ publique De´ mocratique du Congo 4 Department of Biological Sciences, Columbia University, New York, NY, USA

Keywords Abstract allopolyploid evolution; Albertine Rift; whole genome duplication; advertisement calls; We describe a new octoploid species of African clawed frog (Xenopus) from the DNA barcode. Lendu Plateau in the northern Albertine Rift of eastern Democratic Republic of the Congo. This species is the sister taxon of Xenopus vestitus (another octoploid), Correspondence but is distinguished by a unique morphology, vocalization and molecular diver- Ben J. Evans, Department of Biology, Life gence in mitochondrial and autosomal DNA. Using a comprehensive genetic Sciences Building Room 328, 1280 Main sample, we provide new information on the species ranges and intra-speciﬁc Street West, McMaster University, diversity of African clawed frogs from the Albertine Rift, including the details of Hamilton, ON, Canada L8S 4K1. Tel: +1 a small range extension for the critically endangered Xenopus itombwensis and 905 525 9140 Ext. 26973; Fax: +1 905 522 previously uncharacterized variation in Xenopus laevis. We also detail a new 6066 method for generating cytogenetic preparations in the ﬁeld that can be stored at Email: [email protected] room temperature for up to 3 weeks. While extending our understanding of the extant diversity in the Albertine Rift, this new species highlights components of Editor: Mark-Oliver Rodel¨ species diversity in ancestral African clawed frogs that are not represented by known extant descendants. Received 13 June 2010; revised 5 October 2010; accepted 5 October 2010 doi:10.1111/j.1469-7998.2010.00769.x

Introduction species in the laboratory (Kobel & Du Pasquier, 1986; Kobel, 1996). African clawed frogs in the genus Xenopus have a complex Phylogenetic relationships among extant African clawed evolutionary history characterized by both bifurcating and frogs (Xenopus and Silurana) suggest the previous existence reticulating modes of speciation (reviewed in Evans, 2008). of up to three ancestral diploids and three ancestral tetra- Described species include eight tetraploids, six octoploids ploid species that are not represented by a known extant and two dodecaploids; additional undescribed species species with the same ploidy level (Evans, 2007, 2008). Even have been inferred using molecular markers (Evans et al., though these species may now be extinct, the genomes of 2004; Evans et al., 2005; Evans, 2007). Some polyploid these ancestors persist as a portion of the genomes of extant species, such as the octoploids Xenopus itombwensis and allopolyploid species. The prediction that these ‘lost’ ances- Xenopus wittei, are sister taxa that probably evolved by tral species once existed provides motivation for ﬁeld studies ‘regular’ bifurcating speciation from an octoploid ancestor. aimed at discovering their equivalent-ploidy-level descen- Other species pairs, such as the octoploids Xenopus andrei dants, if they exist. and Xenopus vestitus, share recent common ancestry of In an ongoing effort to understand the evolution and half of their genomes while the other half is more divergent. diversity of clawed frogs, we performed ﬁeld collections and This observation is best explained by allopolyploid molecular analyses in regions where allopolyploid descen- speciation – speciation by genome duplication that is asso- dants of the predicted tetraploid ancestors currently occur, ciated with hybridization among species. This mode of including the Albertine Rift of Central Africa. This effort speciation is further suggested by the creation of allopoly- recently resulted in the discovery of the octoploid species X. ploid individuals by hybridizing and backcrossing different itombwensis (Evans et al., 2008) and subsequent searches

276 Journal of Zoology 283 (2011) 276–290 c 2010 The Authors. Journal of Zoology c 2010 The Zoological Society of London B. J. Evans et al. A new species of African Clawed frog (Xenopus) identified another new species that is described here. The parameters, multiple calls from at least two individuals were inferred evolutionary relationships between this new species, analyzed. To communicate information about inter- and which is octoploid, and other extant species further support intra-individual variation in the vocal parameters, we report the notion that tetraploid ‘lost’ ancestors once existed. the grand mean and standard deviation across all replicates, and the sum of squares and residuals divided by their respective degrees of freedom, which is the number of indivi- Methods duals minus one for comparisons among individuals or the number of calls minus one for comparisons among calls. Field collection Advertisement calls were evoked by priming males with The new species was first collected by E. G. and C. K. in July human chorionic gonadotropin (Sigma, Oakville, ON, 2009 on the Lendu Plateau, northern Albertine Rift, Demo- Canada; 100 international units). Vocalizations were recorded cratic Republic of the Congo (DRC), and molecular analyses 6 h after the injection, at the start of the night cycle. An prompted a subsequent expedition by B. J. E. and C. K. to this injected male and a sexually unreceptive female were placed locality in December 2009, in order to further characterize this in a 75.7 L aquarium, two-thirds filled with unchlorinated 1 species in terms of its morphology, distribution and vocaliza- water at 20 C. Recordings were made with a hydrophone (HighTech Inc.; Gulfport, MS, USA sensitivity=164.5 tion, and to provide additional voucher specimens. Other 1 samples in this study were collected by E. G., C. K., B. J. E. dB re 1 V uPa ), stored on CDs (Marantz, Mahawah, NJ, and colleagues, or are reported previously (Evans et al., 2004). USA; model CDR300) and analyzed using SIGNAL software version 5.0 (Engineering Design, Berkeley, CA, USA). To provide information on comparative morphology, we measured 12 external morphological characteristics follow- Analysis of male vocalization and morphology ing Tinsley (1973) from preserved specimens of the new Male clawed frogs produce species-specific advertisement species and of a sympatric population of Xenopus laevis. vocalizations (calls) by opening a pair of cartilaginous disks Additional information on specimens examined is available in the larynx to produce a ‘click’ (Yager, 1992). Calls of in the supporting information. some species are composed of only one click, whereas others produce trains of clicks called a trill. In this study, we Cytogenetics recorded advertisement calls from four males from the new species and compared these with a previous analysis of calls We previously reported a new method to obtain Xenopus from X. itombwensis, X. vestitus and X. wittei (Evans et al., cytogenetic preparations by directly harvesting and fixing 2008) with data from two additional calls from X. wittei. liver tissue (Evans et al., 2008). Here, we adapted this Based on analyses discussed below, these species are closely method to allow the cells to be harvested and fixed in the related to the new species. We report vocal parameters from field, and stored at room temperature for up to 3 weeks only two individuals from the new species because the during transport back to the laboratory. Animals were intervals between clicks of the other two were not suffi- killed by immersion in an 1% MS222 (Sigma) solution. ciently distinguishable for individual clicks to be measured. The liver was accessed through a lateral incision and an We measured call duration, the number of clicks per call, 8mm3 piece was placed on a pre-cleaned glass slide, wetted the inter-click interval (time between the start of one click with three to four drops of 0.04 M KCl and minced with a and the start of the next click), the intensity modulation (the new razor blade. The resulting tissue/KCl slurry was then change in intensity between the softest and the loudest clicks transferred to a 15 mL centrifuge tube and 0.04 M KCl calculated as [(max–min)/min], the call duty cycle (the solution was added to produce a final volume of 13.5 mL. proportion of vocalization in a bout of calls), and the two The mixture was gently inverted/agitated and incubated at sound frequencies with the most energy (dominant frequen- room temperature for 1 h with occasional agitation and cies; DFlow is the lower and DFhigh is the higher of the two inversion. During this time, a fresh 3:1 methanol:glacial frequencies) and inter-call interval (time between successive acetic acid fixative was prepared. After a final agitation, the calls in a bout of calling). Most of these parameters have a tissue/KCl mixture was left for 2 min to allow debris to settle single value for each call. The inter-click interval is a mean to the bottom of the tube. Ten millilitres was drawn from the value calculated from all inter-click intervals in a call. Call top and transferred to a new 50 mL tube. The remaining cell duty cycle and inter-call interval are measured from bouts of suspension and tissue debris were discarded. Two mL of the advertisement calls. Advertisement calls, or bouts of adver- fixative were added to the 50 mL tube, followed by gentle tisement calls, were randomly selected and analyzed. Evans agitation. An additional 10 mL of fixative was then added, et al. (2008) ranked the dominant frequencies by amplitude followed by gentle agitation. The tube was then topped off and presented the mean across individuals of the highest and with fixative to a total of 50 mL, capped and gently agitated. second-highest frequencies. Here, we instead rank the two At this point, the samples were stored at room temperature frequencies with the highest amplitude by frequency and for up to 3 weeks until they were transported back to the report the mean of these frequencies (DFlow and DFhigh). laboratory. This is more appropriate because the two frequencies vary in In the laboratory, 5–12 mL of the field-fixed samples were relative amplitude from call to call. For almost all of these transferred to a 15 mL tube, topped up to 13 mL with a fresh

Journal of Zoology 283 (2011) 276–290 c 2010 The Authors. Journal of Zoology c 2010 The Zoological Society of London 277 A new species of African Clawed frog (Xenopus) B. J. Evans et al.

3:1 fixative and washed four times by centrifugation at 500 g of 500 000 and 100 000 generations (mtDNA and RAG1 for 8 min. For each wash, the supernatant was removed by datasets, respectively) was discarded before the construction aspiration down to 0.25 mL and resuspended in 13 mL of a of a consensus topology based on plots of the posterior fresh fixative by vortexing. In the final resuspension, 0.5–3 distribution of tree likelihoods and parameter estimates. mL of fixative was then added to yield a suitable concentra- tion for slide making. Cleaned slides were wetted with a Species concept drop of 3:1 fixative and left to almost dry before a drop of cell suspension was allowed to fall on the surface from a Essentially all species concepts share a common theme of height of 10 cm. After 7 s, an additional drop of 3:1 referring to evolutionarily distinct lineages that persist fixative was allowed to fall onto the slide before it was through time, multiple lines of evidence can be used to moved to a humid environment and allowed to dry com- identify species, and the chronology by which these lines of pletely. Slides were incubated at 90 1C for 1 h, cooled to evidence become diagnostic varies (de Queiroz, 1998). Even room temperature, stained in 8% (v/v) Giemsa in Gurr at a genetic level, the onset of genetic barriers to reproduc- buffer (pH 6.8) for 2–5 min, rinsed three times in milli-Q tion can be a gradual process that occurs at different times in different parts of the genome (Wu, 2001). For these reasons, H2O and dried. Using standard bright-field microscopy, metaphases were located on slides and then captured as we adopt a general lineage concept of species, where a digital images. The remaining cell suspensions were stored at species is defined as a separately evolving lineage of con- 20 1C. nected subpopulations (metapopulations) through time (de Queiroz, 1998, 2007). We provide information on morphological, behavioral and genetic divergence as evidence for Molecular data, phylogenetic analysis lineage separation. We estimated the phylogenetic history of the new species using molecular data from mitochondrial DNA (mtDNA) Taxonomic account and autosomal DNA (aDNA). The mtDNA data included Xenopus lenduensis, new species 819–2924 base pairs (bp) from 12S and 16S ribosomal Lendu Plateau clawed frog DNA, the intervening transfer RNAVal and cytochrome c (Fig. 1) oxidase subunit I (CO1) genes from all known species of African clawed frog, including 2920 or 2921 bp from four samples of the new species. Pipa pipa was used as an Holotype outgroup to African clawed frogs (Roelants & Bossuyt, 2005). Each species in the mtDNA dataset had at least one MCZ A-139853 (field number BJE2957), adult male: Demo- full-length (42900 bp) sequence; shorter sequences were cratic Republic of the Congo, Orientale Province, Bunia included to characterize species’ ranges. The aDNA data District, Djugu Territory, Aboro Region, Aboro West Village, included cloned paralogs from 317–3054 bp of paralogs of 2.01641N 30.83111E, 2081 m a.s.l. December 8, 2009, collected the RAG1 from all known species of African clawed frogs, by B. J. E. and C. K. including 1141 bp from three paralogs from one individual of the new species. MtDNA and aDNA were amplified Paratypes using previously published primers and aDNA was cloned using Invitrogen cloning kits (Burlington, ON, Canada) as MCZ A-139855 (field number BJE2945), adult male: Demo- described previously (Evans et al., 2004, 2005; Ivanova, cratic Republic of the Congo, Orientale Province, Bunia deWaard & Hebert, 2006; Evans, 2007). Data are deposited District, Djugu Territory, Jiba Region, Goki Lake, near in Genbank (accession numbers are in supporting informa- Jiba, 1.86881N, 30.69591E, 1835 m a.s.l. December 11, 2009, tion Table S1 and Evans et al., 2004, 2008; Evans, 2007). collected by B. J. E. and C. K. Phylogenetic relationships among mtDNA sequences MCZ A-139856 (field number BJE2951), other informa- were estimated using MrBayes version 3.1.2 (Huelsenbeck tion the same as MCZ A-139855 except collection site is at & Ronquist, 2001) using a partitioned analysis with a stem- 1.86041N, 30.68801E, 1847 m a.s.l. loop (doublet) model for the rDNA genes and a general MCZ A-139854 (field number BJE2958), adult female: time-reversible model with a proportion of invariant sites Democratic Republic of the Congo, Orientale Province, and a gamma-distributed rate heterogeneity parameter Bunia District, Djugu Territory, Aboro Region, Ngonse- (GTR+I+G) for the CO1 sequence, which was selected neyu Town 1.98511N, 30.81181E, 1979 m a.s.l. December 6, using the Akaike Information Criterion with MrModeltest 2009, collected by B. J. E. and C. K. (Nylander, 2004). The RAG1 phylogeny was also estimated UTEP20186 (field number EBG2343), adult male: using MrBayes with partitioning by codon position and a Democratic Republic of the Congo, Orientale Province, GTR+I+G model for each position, with model selection Bunia District, Djugu Territory, Aboro Region, 2.00871N, using Bayes factors described in Evans (2007). Both analyses 30.83521E, 2065 m a.s.l. July 6, 2009, collected by E. G used Dirichlet priors for the rate matrix. Two independent and C. K. MCMC runs were performed for 2.6 and 1.8 million UTEP20192 (field number EBG2350), adult female: other generations for mtDNA and RAG1, respectively. A burnin information the same as UTEP20186.

278 Journal of Zoology 283 (2011) 276–290 c 2010 The Authors. Journal of Zoology c 2010 The Zoological Society of London B. J. Evans et al. A new species of African Clawed frog (Xenopus)

common ancestor, it is nonetheless descriptive because it does include all descendants of one of the inferred tetraploid ancestors – ancestor ‘B.’ As detailed below, Xenopus lenduensis can be distinguished from other members of the vestitus-wittei subgroup and other clawed frogs by: (1) its range on the Lendu Plateau; the only other African clawed frog in this region is X. laevis; (2) a unique combination of morphological characters including the absence of a metatarsal claw, short toes, variable presence of dorsal and ventral patterning, a small- to medium-sized subocular tentacle and a rounded snout; (3) its male advertisement call, which is shorter than other members of the vestitus- wittei group and characterized by several unique spectral characteristics not found in other clawed frog species; (4) divergent mitochondrial and autosomal genes.

Morphology and variation Xenopus lenduensis is a medium-sized African clawed frog with the snout–vent length (SVL) of females and males averaging 48 and 40 mm, respectively (Table 1). This species has three claws, a prominent but small metatarsal tubercle with no claw, and a subocular tentacle that sometimes extends beyond the margin of the head when viewed from above. Snout shape is variable but generally more rounded than triangular, and dorsal coloration of X. lenduensis is light green to olive (Figs 1 and 2a–p). Darker olive spots are usually present on the dorsum, variable in size from 0.5 to 3 mm, sometimes more densely distributed caudally, or instead present as larger dark olive and irregularly shaped patches. Some individuals have dark brown or black spots on the dorsum, dark olive triangular patches sometimes posterior to the eyes and/or a dark brown patch between the nostrils and the eyes (Fig. 2d–h). Some individuals have a light band connecting the eyes. One individual was observed with a dark olive hood over the dorsal surface of the head, similar to the commonly observed condition in X. vestitus (Tinsley, 1973). Some individuals of the new species have a light olive hood similartothatvariablyreportedinX. wittei (Tinsley et al., 1979). Nuptial pads of X. lenduensis (when present, males Figure 1 Pictures of holotype MCZ A-139853, (ﬁeld number BJE2957) only) extend from the base of the arm to the tips of the ﬁngers. including (a) dorsal view in life, (b) ventral view immediately after The ventral surface of the legs of X. lenduensis is orange death, (c) dorsal and (d) ventral view after preservation, and close-ups or yellow, with this color sometimes extending anteriorly after preservation indicated by arrows of the (e) subocular tentacle over the ventral surface of the body and ventral surfaces of and (f) metatarsal tubercle. (b) Red coloration on the left side, snout the arms and head, or fading to a dark creamy color over the and neck of the animal is blood from the incision made for genetic ventral surface of the body and head, or with a fairly abrupt sampling, which appears as a maroon ‘J’ on the left side of the vent transition from orange to cream in the pelvic region (Fig. just below the attachment point of the arm. 2o). The legs and ventral surface sometimes have a few light spots (Fig. 2i) but more commonly with lesser pigmented patches or spots, which are sometimes pink or purple, and Diagnosis sometimes less or more dense on the medial portion of the Evolutionary analyses discussed below indicate that X. legs and belly (Fig. 2j–p). Like X. vestitus (Tinsley, 1973), the lenduensis is a member of the vestitus-wittei subgroup of digits are relatively short (Table 1). African clawed frogs (Kobel, Loumont & Tinsley, 1996), which includes X. itombwensis (Evans et al., 2008), X. wittei (Tinsley, Kobel & Fischberg, 1979) and X. vestitus (Laurent, Comparison of morphology with other species 1972; Tinsley, 1973). While this group is not a clade because Xenopus lenduensis can be distinguished from species in the it does not include all descendants of the groups’ most recent genus Silurana, and from Xenopus amieti, X. andrei, Xenopus

Journal of Zoology 283 (2011) 276–290 c 2010 The Authors. Journal of Zoology c 2010 The Zoological Society of London 279 A new species of African Clawed frog (Xenopus) B. J. Evans et al.

Table 1 Morphological measurements of African-clawed frogs from the Lendu Plateau and proximal regions, Democratic Republic of the Congo, including the holotype of Xenopus lenduensis Xenopus lenduensis Xenopus laevis Females; n=27 Males; n=15a Females; n=9 Males; n=14 Measurement Holotype Average StDev Max Average StDev Max Average StDev Max Average StDev Max Body length 37.69 48.10 5.21 56.43 40.25 2.76 46.02 63.19 4.41 67.75 49.94 2.67 57.41 Body width 15.39 22.01 4.03 28.26 16.50 1.39 18.60 29.58 4.04 36.28 19.80 1.22 22.42 Head width 8.43 9.50 0.72 11.02 8.42 0.69 10.07 12.34 1.01 13.36 10.55 0.41 11.09 Snout length 2.9 3.63 0.47 4.84 3.10 0.34 3.93 4.87 0.60 5.65 4.42 0.40 5.14 Eye diameter 1.78 1.88 0.18 2.24 1.64 0.22 2.01 2.76 0.42 3.27 2.49 0.19 2.77 Interocular distance 5.22 4.64 0.38 5.39 4.21 0.34 5.22 6.15 0.47 6.67 4.81 0.72 5.59 Nostril diameter 1.62 1.42 0.21 1.91 1.29 0.20 1.62 1.61 0.21 1.98 1.42 0.18 1.85 Internarial distance 1.98 1.91 0.21 2.61 1.67 0.29 2.09 2.50 0.33 3.02 2.31 0.36 3.30 Tibia length 16.61 18.33 1.42 20.48 16.69 1.10 18.42 23.84 2.21 28.05 20.92 0.91 22.33 Fifth Toe length 12.66 15.04 1.68 17.64 13.36 1.62 17.29 22.69 2.39 25.05 19.41 1.12 20.87 Elbow-first finger 14.62 15.23 1.00 16.93 14.57 0.66 16.30 21.48 2.24 24.77 18.54 0.78 19.70 First finger length 4.3 4.63 0.49 5.64 4.35 0.44 5.09 7.96 1.48 9.89 6.40 0.70 7.50 aIncluding the holotype. boumbaensis, Xenopus clivii, Xenopus fraseri, Xenopus pyg- bronze to silvery bronze luster (Tinsley, 1973; Channing & maeus and Xenopus ruwenzoriensis by the absence of a claw Howell, 2006). Dorsal coloration of X. wittei has been on the metatarsal tubercle in X. lenduensis. Xenopus lenduen- reported to be a uniform gray–green, yellow–green or olive sis can be distinguished from Xenopus gilli and Xenopus without prominent markings or spots (Tinsley et al., 1979; largeni by the presence of a subocular tentacle in X. Channing & Howell, 2006), which would be readily distin- lenduensis, and by unique dorsal longitudinal blotches pre- guished from the usually mottled dorsal pattern of X. sent in X. gilli but absent in other species of African clawed lenduensis. However, we collected and sequenced X. wittei frog, including X. lenduensis. Xenopus lenduensis can be individuals from Kahuzi-Biega National Park (eastern distinguished from X. laevis, Xenopus borealis, Xenopus DRC) that had some dorsal markings that are similar to X. muelleri and X. new tetraploid 1 (Evans et al., 2004) by the lenduensis. For this reason, it is difficult to distinguish size of the eyes (Tinsley et al., 1979), which are much smaller Kahuzi-Biega populations of X. wittei from X. lenduensis in the new species. Xenopus lenduensis can be distinguished based on dorsal coloration alone. Xenopus lenduensis does from Xenopus longipes by the comparatively short digits of not have a dark dorsal band situated caudally with respect to X. lenduensis – for example, the fifth toe is shorter than the the eyes that is variably observed in X. itombwensis (Evans tibia length in X. lenduensis (Table 1), but longer than the et al., 2008). Xenopus lenduensis occasionally has ventral tibia in X. longipes (Kobel et al., 1996). patterning of very dense spots (Fig. 2l) that is variably Distinguishing X. lenduensis from other species in the observed in X. vestitus (Tinsley, 1973) and X. wittei (Tinsley vestitus-wittei subgroup based on morphology is best et al., 1979), although the ventrum of X. lenduensis usually achieved with a combination of other data on provenance, has sparse spots, such as on the holotype (Fig. 1c). A key vocalization and/or molecular variation. The size of X. difference between X. vestitus and X. wittei is the distance lenduensis is similar to X. wittei and X. vestitus, which have between the eyes (Tinsley et al., 1979); the eyes of X. vestitus average adult female SVL typically 46 and 47 mm, respec- are relatively large and usually more than half of the inter- tively, and a male SVL of 37 and 38 mm, respectively ocular distance (Tinsley, 1975), whereas the eyes of X. wittei (Tinsley et al., 1979; Kobel et al., 1996). Xenopus itombwen- are comparatively smaller and the inter-ocular distance is sis is slightly smaller than these species, with female SVL about 2.2 times the eye diameter (Tinsley et al., 1979). averaging 35 mm and male SVL around 30 mm, although Surprisingly, the spacing and size of the eyes of X. lenduensis this may be an underestimate of the typical size of adults are more similar to X. wittei than to its sister taxon X. (Evans et al., 2008). Dorsal coloration of X. vestitus is a vestitus; the inter-ocular distance averages 2.5 (females) or marbled pattern of light silver-golden to bronze chromato- 2.6 (males) times the eye diameter (Table 1). Another phores over a brown background, sometimes with a dark (related) difference is the shape of the snout, which tends to transverse collar at the base of the head, a light post-orbital be more pointed in X. vestitus than in X. itombwensis and X. area or a light cephalic hood (Tinsley, 1973; Tinsley et al., wittei (Tinsley et al., 1979; Evans et al., 2008). The snout of 1979; Kobel et al., 1996), a pattern that is different from that the new species tends to be rounded (Fig. 2). generally observed in X. lenduensis. The dorsal surface of X. Xenopus lenduensis is readily distinguished by morphol- lenduensis sometimes has dark brown spots, such as in the ogy from X. laevis, a species with which it is often found (in holotype (Fig. 1a), whereas the dorsal surface of X. vestitus the same ponds). Xenopus laevis is significantly larger in size lacks dark spots because flecks of darker pigments are finely than X. lenduensis (Po0.0001 for comparisons with each intermingled with flecks of lighter pigments, imparting a sex, Student’s t-test, Table 1) and has significantly larger

280 Journal of Zoology 283 (2011) 276–290 c 2010 The Authors. Journal of Zoology c 2010 The Zoological Society of London B. J. Evans et al. A new species of African Clawed frog (Xenopus)

(a) (b) (c) (d)

(e) (f) (g) (h)

(i) (j) (k) (l)

(m) (n) (o) (p)

Figure 2 Morphological variation of Xenopus lenduensis in life and after preservation. Dorsal views include preserved specimens: (a) paratype MCZ A-139856 (field number BJE2951), (b) paratype MCZ A-139854 (field number BJE2958), (c) paratype MCZ A-139855 (field number BJE2945) and live unvouchered individuals (d–h). Ventral views include preserved specimens: (i) UTEP20198 (field number EBG2356), (j) paratype MCZ A- 139856, (k) paratype MCZ A-139855, and (l) paratype MCZ A-139854 and live unvouchered individuals (m–p). eyes, even after dividing by body size (Po0.001, Po0.0001 Vocalization for comparisons between females and males, respectively, Student’s t-test). Xenopus laevis is also slimier in the hand The male advertisement call of X. lenduensis is a brief, rapid, than X. lenduensis, possibly due to more viscous and plenti- trill with short intervals between calls (Table 2, Fig. 3). The ful skin secretions and/or its larger size. interval between clicks is so short (12 milliseconds) that individual clicks are often difficult to distinguish. The inter-call interval is much shorter in X. lenduensis than X. vestitus and X. wittei. Another major difference between the vocalization of Size dimorphism thenewspeciesanditsclosestrelativesisthemuchshorter Females are significantly larger than males (Po0.0001, duration of the X. lenduensis call. The dominant frequencies of Student’s t-test, Table 1), which is consistent with other X. lenduensis are very similar to X. vestitus, but not X. species of African clawed frog (Kobel et al., 1996). itombwensis or X. wittei (Table 2). The call of X. lenduensis

Journal of Zoology 283 (2011) 276–290 c 2010 The Authors. Journal of Zoology c 2010 The Zoological Society of London 281 A new species of African Clawed frog (Xenopus) B. J. Evans et al.

Table 2 Advertisement call characteristics of Xenopus lenduensis, Xenopus vestitus, Xenopus wittei and Xenopus itombwensis X. lenduensis CN Duration IM IclickI DF (low) DF (high) CDC IcallI Grand mean 9.84 143.86 13.25 11.97 1608.62 2148.01 0.89 169.71 stdev 0.97 9.70 4.37 1.04 24.20 20.77 0.04 14.86 SSbetween(1) 2.86 1321.23 127.10 16.30 20936.60 15288.70 0.00 4008.80 Residuals(36) 0.89 60.08 16.13 0.66 20.40 18.70 0.00a 115.40 X. vestitus Grand mean 89.75 1167.02 35.69 12.72 1595.64 2154.50 0.25 1918.52 stdev 16.92 220.17 23.00 0.36 5.80 9.71 0.04 658.82 SSbetween(1) 132.25 7425.00 72.84 0.10 19.13 5.55 0.00 436065.00 Residuals(2) 363.25 68997.00 756.81 0.14 40.87 138.67 0.00b 433542.00b X. wittei Grand mean 45.20 1397.10 6.74 31.06 1288.78 1521.97 0.64 4995.63 stdev 17.60 486.28 5.24 4.18 33.55 32.44 0.24 4553.92 SSbetween(1) 90.13 8.00 70.62 69.01 4480.40 4043.80 0.00 9567602.00 Residuals(3) 382.89 315293.00 13.03 0.34 7.00 55.40 0.08 21536114.00c X. itombwensis (fast trill) Grand mean 18.82 326.20 4.89 17.57 1284.16 1536.69 0.88 681.08 stdev 3.84 59.08 2.44 2.47 174.21 165.20 0.09 102.02 SSbetween(1) 5.60 713.00 35.57 4.24 56202.00 25232.00 0.01 49067.00 Residuals(9) 15.78 3800.00 2.68 6.33 27477.00 27518.00 0.01d 7186.00e X. itombwensis (slow trill) Grand mean 8.82 326.66 0.91 36.43 1145.73 1644.96a –– stdev 2.40 101.09 0.84 9.55 141.36 11.37 – – SSbetween(1) 3.10 9412.00 2.35 9.22 160590.00 – – – Residuals(9) 6.06 10309.00 0.43f 100.39 2406.00g –– –

Mean, standard deviation (stdev), sum of squares between individuals divided by the degrees of freedom (SSbetween), and residuals divided by the degrees of freedom (Residuals) are provided for call duration in milliseconds (duration), number of clicks per call (CN), intensity modulation (IM), interclick interval in milliseconds (ICI), two dominant frequencies in kiloHertz (DFlow and DFhigh), and call duty cycle (CDC). See ‘Methods’ for more information on these parameters. Measurements of the fast and slow trill portions of the X. itombwensis advertisement call are indicated separately. For X. lenduensis, results are sumarized for two individuals for three calls per individual. One hundred and twenty-three and 251 clicks were analysed, respectively, for each individual. For SSbetween and Residuals degrees of freedom are indicated in parentheses by row except where noted, or not indicated when data were obtained from only one individual, or not indicated for CDC for the slow trill of X. itombwensis because CDC is a parameter calculated from both portions of the call. aFour measurements from one individual. b4 d.f. c14 d.f. d13 d.f. e12 d.f. f6 d.f. g8 d.f. lacks two distinct phases that characterize the call of X. present, becoming denser caudally and on the dorsal surface of itombwensis and some other species such as X. laevis (Kobel the toes, less dense on the legs. Ventral surface bright orange et al., 1996). A recording of the call of X. lenduensis has been with dense purplish grey stipples under the head and belly, submitted to Amphibiaweb (http://www.amphibiaweb.org). with a clear orange band in the center and with medium-sized purple spots on the ﬂanking region, especially in a ring around Description of the holotype and color of the the insertion point of each arm. Inguinal region and ventral surface of legs and feet orange with larger purple spots on the holotype in life margins of the dorsal surfaces of the legs and feet. Morpholo- Holotype an adult male, small subocular tentacle present, gical measurements of the holotype are listed in Table 1. claws present on toes I–III, metatarsal tubercle prominent but without a claw, webbing complete. Nuptial pads visible on the Color of the holotype in preservative ventral surface of arms extending from the base of arms over the humeral area and forearm. Dorsum olive, with a dark Dorsum dark olive with brown spots over the back and the brown spot on the dorsal surface, 2mm in diameter and dorsal surface of the hind legs, transitioning laterally on positioned caudally with respect to the right eye and anterior ﬂanks to a cream-colored venter; dorsal surface of head to the arms (Fig. 1a). Smaller lighter brown spots variably dark olive; dorsal surface of limbs olive with dark olive spots

282 Journal of Zoology 283 (2011) 276–290 c 2010 The Authors. Journal of Zoology c 2010 The Zoological Society of London B. J. Evans et al. A new species of African Clawed frog (Xenopus)

Figure 3 The male vocalization of (a) Xenopus lenduensis is distinct from that of closely related species (b) Xenopus vestitus, (c) Xenopus wittei and (d) Xenopus itombwensis. A bout of four advertisement calls is shown for X. lenduensis; a single advertisement call is shown for the other species. with a high density of spots on toes; ventral side of the head Ecology and distribution speckled with small dark olive spots; venter cream over a central band with fairly dense brown spots on lateral sides of Xenopus lenduensis was collected from multiple locations on venter; ventral surface of the hind feet cream with some brown the Lendu Plateau, northern Albertine Rift, DRC (support- spots near the margin with the dorsal surface on upper legs. ing information Table S1; Fig. 5). Xenopus lenduensis was present in small populations of o40 individuals in small bodies of standing water that were occasionally encountered Ploidy in disturbed agricultural areas. When the habitat was highly Xenopus lenduensis is octoploid and has 72 chromosomes disturbed or cleared of vegetation, for example in areas used (Fig. 4). The ploidy level revealed by cytogenetic analysis for water supply by people, this species was not observed, was consistent with ploidy inferences based on phylogenetic although X. laevis was present. Xenopus lenduensis therefore analysis of divergent paralogs of the recombination activat- seemed to be detrimentally impacted by habitat modiﬁca- ing gene 1 (RAG1). tion. The Lendu Plateau was probably once a savannah forest mosaic, with forests occurring in patches above 1500 m (Fishpool & Collar, 2006). In the areas where we Xenopus lenduensis barcode collected the new species, the forest has been completely The Genbank accession number of the holotype barcode destroyed; the plateau is now essentially entirely grassland based on the sequence from the CO1 gene (Hebert et al., with a few isolated trees, many of which are introduced. We 2003) is HQ225707. did not see any primary or secondary forest during our

Journal of Zoology 283 (2011) 276–290 c 2010 The Authors. Journal of Zoology c 2010 The Zoological Society of London 283 A new species of African Clawed frog (Xenopus) B. J. Evans et al.

species contributed their genomes to extant allo-octoploid species such as X. lenduensis, but are not represented by a known extant representative with the same ploidy level (Evans, 2007, 2008). In Fig. 6, these inferred tetraploid ancestors are labeled‘A’,‘B’and‘C.’Byfocusing on nodes with at least 95% posterior probabilities in the mtDNA and aDNA trees to infer ancient instances of allopolyploidization, at least three independent allooctoploidization events are suggested. These events include the following pairs of tetraploid ancestors (with the maternal tetraploid ancestor listed first): B+Agaverisetoan ancestor of X. lenduensis and X. vestitus,C+Agaverisetoan ancestor of X. amieti, X. andrei, X. boumbaensis, X. longipes, X. ruwenzoriensis and X.cf.boumbaensis and C+Bgaverisetoan ancestor of X. itombwensis and X. wittei (Fig. 6). This inference is consistent with those of Evans (2007), which did not include data from X. lenduensis. Interestingly, X. lenduensis RAG1 paralog b1 has three frameshift mutations, whereas RAG1 paralog a1 and RAG1 paralog a2 appear to be functional at the coding level. This provides additional support for the finding of Evans (2007) that pseudogenization of RAG1 paralogs occurred in a Figure 4 Giemsa-stained metaphase stage cell from an Xenopus lenduensis illustrating octoploidy (8n=72 chromosomes). This indivi- phylogenetically biased fashion, such that RAG1 paralogs dual was sampled in Ngensengeyu (DRC), MCZ A-140087; fieldcode inherited from one diploid ancestor (the ‘b’ ancestor) be- BJE2929. came pseudogenes more frequently than paralogs inherited from the other diploid ancestor (the ‘a’ ancestor). surveys; dense weedy vegetation was sometimes observed in small, highly disturbed valleys. Etymology The new species is named after the plateau where it occurs – the Lendu Plateau of the Albertine Rift in Orientale Pro- Evolutionary relationships with other African vince, DRC, which lies just west of Lake Albert. This region clawed frogs is also known as the Blue Mountains. Independent analyses of unlinked genomic regions suggest with strong support that X. lenduensis and X. vestitus are African clawed frog mtDNA phylogeography in the sister species derived from the same allo-octoploid ancestor. Albertine Rift mtDNA of these species places them together in a strongly supported clade (Fig. 5). Using the full mtDNA dataset, the With the exception of X. laevis, no species we sampled had uncorrected p-distance between X. lenduensis and X. vestitus substantial levels of intra-specific mtDNA divergence in the is 0.04048. The uncorrected p-distance between these species Albertine Rift. Our analysis slightly extends the known including only the COI ‘barcode’ region is 0.09386. The range of the critically endangered X. itombwensis to prox- uncorrected p-distance between these species including only imate localities on the Itombwe Plateau. This species was the 16S fragment proposed as a barcode fragment for formerly known only from its type locality, whose GPS amphibians minus 78 bp on the 30 end (Vences et al., 2005) is coordinates were incorrectly reported in the species descrip- 0.03766. This mtDNA relationship supports the previous tion, but are correctly reported here (supporting informa- existence of a maternal (i.e. mtDNA-contributing) tetraploid tion Table S1; Evans et al., 2008). Because the range ancestor of this pair of octoploid species. In Figs 5 and 6, this extension here is so small, this does not warrant changing tetraploid ancestor is indicated by lineages labeled ‘B.’ the conservation status of this species. The Itombwe Plateau We cloned alleles from three of the four expected RAG1 has the most threatened species of amphibians and the paralogs from X. lenduensis, and each has a close relationship highest number of endemic amphibians in the Albertine with an orthologous paralog of X. vestitus (Fig. 6). Data were Rift; this is also the case for birds (Plumptre et al., 2007). not obtained from RAG1 paralog b2fromX. lenduensis.Thisis Our analysis indicates that X. ruwenzoriensis also occurs consistent with an absence of data from closely related paralogs in the DRC (Fig. 5; Greenbaum & Kusamba, 2010); this of X. amieti, X. andrei, X. longipes, X. ruwenzoriensis and X.cf. species was formerly only known from the type locality near boumbaensis (Evans, 2007), and could be a result of deletion in Bundibugyo in the Semliki Valley, western Uganda (Fisch- an ancestor. Data from RAG1 paralog b3ofX.cf.boumbaensis berg & Kobel, 1978), and from the Budongo Forest, east of were not obtained, which could be due to deletion or poor Lake Albert, also in western Uganda (IUCN, 2010). amplification (Evans, 2007). Relationships based on aDNA are We identified three diverged mtDNA lineages in X. laevis in consistent with previous inferences that at least three tetraploid the Albertine Rift (Fig. 5). One is closely related to mtDNA

284 Journal of Zoology 283 (2011) 276–290 c 2010 The Authors. Journal of Zoology c 2010 The Zoological Society of London B. J. Evans et al. A new species of African Clawed frog (Xenopus)

Figure 5 Mitochondrial DNA phylogeny and location of samples from the Albertine Rift (insert). In the phylogeny, black dots indicate nodes with Z95% posterior probability. For clarity, some of these nodes are not indicated on terminal branches. At least three tetraploid ancestors are inferred and labeled A, B and C; different combinations of the genomes of these ancestors gave rise to extant octoploid and dodecaploid species (see text). Species with ranges in the Albertine Rift have red names on the phylogeny and symbols indicate their geographic location on the insert. On the insert, regions greater than 2000 m a.s.l are indicated in gray; lakes are blue. lineages sampled from X. laevis populations in East Africa phology (Tinsley, Loumont & Kobel, 1996). Xenopus muelleri (Uganda, Rwanda, Tanzania) and another was closely related occurs over much of east and south-east Africa, extending up to mtDNA lineages sampled in X. laevis populations in Lake Tanganika to the xeric area south of Lake Kivu (near portions of west and south-central Africa (Cameroon, Nigeria, Bukavu). The distribution of X. pygmaeus over much of Zambia, Botswana). The third X. laevis mtDNA lineage from Central Africa reaches its easternmost limit at the region west the Albertine Rift is only known from the Lendu Plateau, and of lakes Kivu (data not shown) and Albert. The distribution to our knowledge, has not been reported previously. of X. wittei was conﬁrmed to include portions of south- Other inferences of species ranges are generally consistent western Uganda, western Rwanda and the Kahuzi-Biega and with previous species distributions that were based on mor- Lubero Highlands in DRC (Tinsley et al., 1979; Kobel et al.,

Journal of Zoology 283 (2011) 276–290 c 2010 The Authors. Journal of Zoology c 2010 The Zoological Society of London 285 A new species of African Clawed frog (Xenopus) B. J. Evans et al.

Figure 6 RAG1 phylogeny including Xenopus lenduensis supports a sister relationship with the octoploid species Xenopus vestitus.Nodes with posterior probabilities equal to 90–95% or 495% are indicated with open and solid dots, respectively. Lettered nodes correspond with each of the two paralogs from the inferred tetraploid ancestors labeled in Fig. 5. Lineages labeled ‘A’ correspond with paralogs from an inferred paternal tetraploid ancestor of X. lenduensis and X. vestitus and another octoploid ancestral species that gave rise to Xenopus amieti, Xenopus andrei and Xenopus boumbaensis, and the dodecaploid species Xenopus longipes, Xenopus ruwenzoriensis and Xenopus cf. boumbaensis. Lineages labeled ‘B’ correspond with paralogs from an inferred maternal tetraploid ancestor of X. lenduensis and X. vestitus, which was also the paternal tetraploid ancestor of Xenopus wittei and Xenopus itombwensis. Lineages labeled ‘C’ correspond with paralogs of an inferred maternal tetraploid ancestor of the octoploid ancestral species that gave rise to X. amieti, X. andrei, X. boumbaensis and X. longipes, X. ruwenzoriensis and X.cf.boumbaensis. An insert depicts a summary of bifurcating and reticulating relationships in clawed frogs with paternally inherited lineages represented by dashed lines and biparentally or maternally inherited lineages represented by solid lines. In the insert, ‘A’, ‘B’ and ‘C’ refer to ancestral tetraploid lineages instead of the ancestral paralogs. Nodes with asterisks refer to putative instances of allopolyploidization.

1996). Xenopus wittei mtDNA sequences from the Kahuzi- to its sister taxon X. vestitus and to X. wittei, but larger than Biega and Lubero highlands are slightly diverged from those X. itombwensis. Dorsal and ventral coloration and morphol- of the type locality in south-western Uganda (Fig. 5). We do ogy distinguish X. lenduensis from its sister taxon X. vestitus, not have additional samples of X. vestitus beyond the one used the former species having an olive dorsum with variable in Evans et al. (2004); other studies report a broader distribu- darker olive spots, a usually rounder snout and more widely tion for this species, but not north of Lake Edward (Tinsley, spaced eyes than X. vestitus. Distinguishing X. lenduensis 1973; Tinsley, 1975). from some populations of X. wittei that have variable spots on the dorsum could be challenging, although other X. wittei Discussion populations lack dorsal spots. The fundamental characteristics of the vocalization of the new species, including call duration and inter-call interval, are distinct from other Distinctiveness of X. lenduensis species of African clawed frog, including X. vestitus and We distinguish a new species of African clawed frog, other members of the ‘vestitus-wittei’ subgroup (Table 2; Xenopus lenduensis, from other species based on morphol- Fig. 2). On a molecular level, mtDNA and nuclear DNA ogy, vocalization and molecular divergence – characteristics divergence between X. lenduensis and X. vestitus is substan- that together identify X. lenduensis under the general lineage tial and comparable to that seen between other closely concept of a species. The most straightforward way to related species of African clawed frog such as X. fraseri and narrow down the identity of the new species is its prove- X. pygmaeus or X. amieti and X. longipes (Figs 5 and 6). nance on the Lendu Plateau, DRC, which is the northern extent of the Albertine Rift. The only other African clawed Allopolyploid speciation and the search for frog that shares this habitat with X. lenduensis is X. laevis. descendants of ‘lost ancestors’ Xenopus lenduensis is easily distinguished from X. laevis by the much larger size and the larger eyes of X. laevis. Within Building on previous molecular phylogenetic studies of the vestitus-wittei subgroup, the new species is similar in size African clawed frogs (Carr, Brothers & Wilson, 1987;

286 Journal of Zoology 283 (2011) 276–290 c 2010 The Authors. Journal of Zoology c 2010 The Zoological Society of London B. J. Evans et al. A new species of African Clawed frog (Xenopus)

Kobel, Barandun & Thiebaud, 1998; Measey & Channing, taxa overlap in the region between Lakes Kivu and Edward 2003; Evans et al., 2004; Evans et al., 2005; Evans, 2007), the in south-western Uganda and the eastern DRC, which is evolutionary relationships between X. lenduensis and other where X. wittei and X. vestitus occur (Tinsley, 1973, 1975; species of clawed frog are evaluated here using mtDNA and Tinsley et al., 1979). Xenopus itombwensis, the sister taxon of sequences from cloned paralogs of the RAG1 gene. X. wittei, occurs on the Itombwe Plateau to the south of Although some paralogs of RAG1 were not sequenced, X. wittei and west of Lake Tanganika. Xenopus lenduensis, possibly because they have been deleted, together, these the sister taxon of X. vestitus, occurs on the Lendu Plateau markers illustrate a sister relationship between X. lenduensis to the north of X. vestitus, on the west side of Lake Albert. and X. vestitus. We speculate that X. lenduensis and X. vestitus are derived from a recent common octoploid ancestor gener- Phylogeography of X. laevis ated from the fusion of two tetraploid genomes indicated by lineages labeled ‘B’ and ‘A’ in Figs 5 and 6. However, we Here, we use ‘X. laevis’ sensu lato following Kobel et al. cannot rule out the less parsimonious possibility that each (1996). This species is distributed from South Africa to species was formed from simultaneous, but independent southern Sudan and from Nigeria to Tanzania. Previous allopolyploidization events involving the same combination studies identified at least six major mtDNA lineages in X. of maternal and paternal tetraploid ancestors. In any case, laevis. These lineages are mostly allopatric, although popu- X. lenduensis and X. vestitus share recent common ancestry of lations with multiple lineages do exist in South Africa (Du one half of their genome with X. itombwensis and X. wittei, Preez et al., 2009). One mtDNA lineage occurs in the Cape and the other half with X. amieti, X. andrei, X. boumbaensis, Region of South Africa south-west and west of the Cape X. longipes, X. ruwenzoriensis and X.cf.boumbaensis. Fold Mountains (Grohovaz, Harley & Fabran, 1996; Evans The discovery of X. lenduensis supports previous work et al., 1997, 2004; Measey & Channing, 2003; Du Preez that inferred the prior existence of ancestral tetraploid et al., 2009). Although subspecies are not recognized here, species that did not leave known extant tetraploid descen- this lineage corresponds in geographic distribution with a dants (Evans et al., 2005, 2008; Evans, 2007). In addition to portion of the ‘laevis’ subspecies (Kobel et al., 1996). A these tetraploid ancestors, one or two ancestral diploid second lineage occurs in central South Africa between the (2N=18) Xenopus species are predicted, depending on Cape Fold Mountains and Lesotho (Du Preez et al., 2009); whether tetraploidization occurred in Xenopus via autopo- this lineage is not included in the analysis presented in Fig. 5. lyploidization or allopolyploidization. In Silurana, analysis A third lineage occurs in Malawi and north-western South of aDNA suggests that tetraploidization occurred by allo- Africa (Grohovaz, Harley & Fabran, 1996; Measey & polyploidization; hence, another diploid (2N=20) ancestral Channing, 2003; Evans et al., 2004; Du Preez et al., 2009). species is predicted to have existed in addition to the A fourth lineage occurs in Congo Brazzaville (Evans et al., ancestor of the diploid species S. tropicalis (Evans et al., 2004). A fifth lineage occurs in north-western and central 2005; Evans, 2007). sub-Saharan Africa in Nigeria, Cameroon, Zambia and the The discovery of descendants of these predicted species DRC, including the southern portion of the Albertine Rift would be an unusual example of unsampled species being (this study; Evans et al., 1997, 2004; Measey & Channing, predicted by phylogenetic inference. The discovery of di- 2003; Du Preez et al., 2009). This lineage corresponds in ploid descendants of the predicted 2N=18 Xenopus diploid geographic distribution with the subspecies ‘sudanensis’ ancestors would be particularly useful for studies of gene and ‘poweri’ (Kobel et al., 1996). A sixth lineage occurs duplication in tetraploid species such as X. laevis. Analysis in East Africa and the central Albertine Rift (this study; of these descendants would allow, for example, the parti- Evans et al., 1997, 2004; Du Preez et al., 2009) and tioning of paralogs and singletons (duplicates where one corresponds in geographic distribution with the ‘victori- copy became a pseudogene) into subgenomes derived from anus’ subspecies (Kobel et al., 1996). Here, we identify a each diploid ancestor. Knowledge of this information would seventh mtDNA lineage within X. laevis that occurs on the allow a large-scale test for evidence of phylogenetically Lendu Plateau and proximate areas west of Lake Albert biased pseudogenization in duplicated genetic networks (Fig. 5). after allopolyploidization. A single-locus example of biased Multiple lines of evidence suggest that X. laevis contains pseudogenization was reported recently in the RAG1 pro- substantially differentiated populations that may warrant teins (Evans, 2007), and this result is further supported here species status (Measey & Channing, 2003). The genetic by the observation that RAG1 paralog b1 is pseudogenized divergence between mtDNA lineages within X. laevis can in X. lenduensis but RAG1 paralogs a1 and a2 seem func- be of a magnitude comparable to that observed between tional at the DNA level. Another example of biased pseu- other species of African clawed frog. For example, in an dogenization in Xenopus is reported by Bewick, Anderson & 870 bp region of the 16S gene, the uncorrected p-distance Evans (2010) at the DMRT1 locus. between the Cape Region population of X. laevis and other The recent discovery of X. lenduensis and X. itombwensis populations is about 3.2%, which is almost as large as is also interesting because it identifies two pairs of sister the divergence over the same region of mtDNA between octoploid species (X. lenduensis+X. vestitus) and (X. X. lenduensis and X. vestitus. Many X. laevis populations itombwensis+X. wittei) that are distributed over much of with divergent mtDNA also have distinct vocalizations the Albertine Rift. The distributions of these pairs of sister (MLT, DBK and BJE unpublished data). It would be

Journal of Zoology 283 (2011) 276–290 c 2010 The Authors. Journal of Zoology c 2010 The Zoological Society of London 287 A new species of African Clawed frog (Xenopus) B. J. Evans et al. interesting to compare these vocalizations with that of X. ably affected patterns of speciation in Central Africa and the laevis from the Lendu Plateau, but unfortunately, we did not Albertine Rift, for example in rodents (Huhndorf, Kerbis obtain recordings of this population. Morphological varia- Peterhans & Loew, 2007), gorillas (Anthony et al., 2007), tion is also substantial – for example, X. laevis from the West birds (Bowie et al., 2006; Voelker, Outlaw & Bowie, 2010) Cape Region, south-west of the Cape Fold Mountains, is and frogs (Evans et al., 2004; Blackburn, 2008). significantly larger than X. laevis from other portions of The recognition of X. lenduensis further characterizes the South Africa (Du Preez et al., 2009). However, analysis of considerable biological diversity in the Albertine Rift, con- mitochondrial and nuclear markers in a transect of indivi- tributing to our growing understanding of amphibian diver- duals running from southwest to north-east South Africa sity in this region (Plumptre et al., 2007; Behangana, identified a zone of admixture (Du Preez et al., 2009). Kasoma & Luiselli, 2009; Greenbaum & Kusamba, 2010 The evolutionary relationships among these lineages have Roelke et al., in press). Unfortunately, the biological diver- implications for elevation of X. laevis populations to species sity of the Lendu Plateau and Albertine Rift faces severe status. According to mtDNA, for example, the X. laevis threats due to a very dense human population (Burgess population from the Cape Region of South Africa is equally et al., 2007) and the associated anthropogenic disturbance diverged from the X. laevis populations from western Africa, that has destroyed natural vegetation on the Lendu Plateau from eastern Africa and from the Lendu Plateau. Additional (B. J. Evans, E. Greenbaum, C. Kusamba pers. obs., Fish- studies are needed to sort out species from population-level pool & Collar, 2006). Based on our locality records, we relationships within X. laevis (Measey & Channing, 2003). estimate the extent of the occurrence of X. lenduensis to be In particular, it would be informative to use genetic data to o5000 km2. Given the catastrophic deforestation and severe explicitly compare a demographic hypothesis of isolation by habitat fragmentation on the Lendu Plateau and the ex- distance within X. laevis sensu lato with a scenario involving pected continuing decline of the extent and quality of this speciation within this clade. habitat, we recommend that this species be classified as endangered under the IUCN Red List Criteria (IUCN, 2001). Biogeography of the Albertine Rift The Great Rift Valley is an 6000 km trench between multiple continental plates. From its northern extent in Acknowledgments Lebanon, it runs south, dividing Jordan and Israel, down We are indebted to Richard Tinsley for providing construc- the east side of the Sinai Peninsula and under the Red Sea. It tive comments on this manuscript, genetic samples of X. surfaces in Eritrea and Ethiopia, dividing the Ethiopian vestitus and many insightful discussions about African highlands, and splits into a Western and an Eastern Rift clawed frogs. We thank Jonathan Dushoff for statistical just north of the equator, around the border between Sudan advice, Adam Bewick and Jonathan Woodward for assis- and Uganda and between Ethiopia and Kenya. The Eastern tance with photography, Jose´Rosado and Joseph Martinez Rift runs through central Kenya and Tanzania and is for curatorial assistance, Miguel Vences and two anon- associated with Mts. Kenya and Kilamanjaro, the highest ymous reviewers for constructive comments on this manu- mountains in Africa. The Western Rift includes the Alber- script and Mwenebatu M. Aristote, Wandege Mastaki tine Rift highlands, which run between the DRC and the Moninga, John and Felix Akuku, Mululema Zigabe, Maur- western borders of Uganda, Rwanda, Burundi and western ice Luhumyo, Jean Marie Chambu, Angalikiya Mulamba Tanzania (Plumptre et al., 2007). Further south, these rifts Marcel and the late Asukulu M’Mema for field assistance. converge in Malawi and continue to central Mozambique. We thank l’Institut Congolais pour la Conservation de la Xenopus lenduensis and its closest relatives are endemic to Nature for our Autorisation de Recherche in DRC, Baluku the Albertine Rift. This area has a distinct flora and fauna Bajope and Muhimanyi Mununu of the Centre de Re- associated with the montane rainforest highlands that cherche en Sciences Naturelles for providing project support grades into lowland tropical rainforests of the Congo Basin and permits. This research was supported by the Canadian to the west and forest savannah mosaic habitat in the east Foundation for Innovation, the National Science and En- and south (Burgess et al., 2004). The Albertine Rift is a high gineering Research Council and the Museum of Compara- priority for biodiversity conservation and is recognized, for tive Zoology at Harvard University. Fieldwork by EG was example, as a biodiversity hotspot (Mittermeier et al., 2004), funded by the Percy Sladen Memorial Fund, the Depart- a distinct terrestrial ecoregion (Burgess et al., 2004), and an ment of Biology at Villanova University, a National Geo- ‘Endemic Bird Area’ (Stattersfield et al., 1998). At least one graphic Research and Exploration Grant (no. 8556-08), an bird subspecies of the White-browed Crombec, Chapin’s IUCN/SSC Amphibian Specialist Group Seed Grant and Crombec (Sylvietta leucophrys chapini), occurs only on the the University of Texas at El Paso. Lendu Plateau – although this subspecies may be extinct due to habitat destruction (Fishpool & Collar, 2006). The exceptional species diversity in this region is in part a References consequence of the varied ecosystems associated with the altitudinal gradient and its location between distinct habitat Anthony, N.M., Johnsin-Bawe, M., Jeffery, K., Clifford, types. Pleistocene and Pliocene climatic oscillations prob- S.L., Abernethy, K.A., Tutin, C.E., Lahm, S.A., White,

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